In the literatures, two terminals of a specific peptide substrate are respectively bound to poly-L-lysine (PL) and fluorochrome. In the other method, two terminals of a peptide substrate are covalently bound to fluorochrome and quencher of the fluorochrome.
Recently, because the developments of hardware and software of optical imaging, the diagnosis of optical imaging improves. In order to enhance the sensitivity and accuracy of the diagnosis of optical imaging, to develop a safe stable targeting probe for optical imaging becomes an important subject of current optical imaging research. A general probe for optical imaging is formed by respectively bonding two terminals of a peptide substrate to a portion of amino acids of poly-L-lysine (PL) and fluorochrome Cy5.5.
Molecular imaging contains magnetic resonance imaging (MRI), nuclear medicine and optical imaging. After the developments in techniques of magnetic resonance imaging, nuclear medicine and optical imaging, in the past ten years, the study and research of in-vivo molecular imaging are rapidly developing and growing. The in-vivo molecular imaging can provide observation and information of an in-vivo biology system at molecular and genetic function level including normal and abnormal cell process. The observation and information are used in the early diagnosis and genomic medicine and rapid development of new medicine of human diseases. The medicines needed by the three imaging instruments shall mainly have biological activity and targeting. For a general conventional medical imaging technique, the conditions of disease during a long-term, such as the size of tumor, are reflected and output only. By using molecular imaging, the short-term biological mutations, such as pre-cancer molecular change, cancer cell short-term transplant, cardiovascular preliminary fibrosis, etc., can be detected. Thus, the molecular imaging is the best early diagnostic method for tumors, coronary disease and chest diseases which threaten the life of human beings. In in-vivo molecular imaging field, the study fields of molecular biology, chemistry, physics, radiotherapeutics, nuclear medicine and computer science are combined because in-vivo imaging can be obtained by contributions of core techniques of each study field. After the recent development of in-vivo molecular imaging was carried out for several years, molecular imaging is superior by comparing to general conventional medical imaging. Many researches were directed to the reaction mechanism and cell composition of disease molecule. The main efforts of them are directed to noninvasive in-vivo imaging technique of high resolution. After the nuclear medicine imaging and magnetic resonance imaging based on human anatomy and pathology are compared to optical imaging, it is known that molecular imaging has entered the threshold of biology molecule calculated by unit of nano meter. Therefore, the early diagnosis and monitor of a disease are studied by gene level, the results and techniques of the study can be intensively applied to biology and disease therapeutics.
Typical fluorochromes use fluorescence having wavelength in visible light range of 400-600 nm. The fluorescence signal of the fluorochromes can be seen by spectrophotometer or fluorescence microscope. But, the photons in this wavelength range are not well used in in-vivo or in-vitro application because tissues and blood will absorb the photons having the wavelength. Recently, fluorescence is used in labeling antibodies, DNA probes, biochemical analogs, lipids, medicaments, cell compositions and polymers. The fluorescence will be suitable for applying in detector and useful as light source after brightness of the fluorescence is enhanced, the light stability of it is increased, the toxicity is decreased, the non-specific bondage is lowered and the excitation and emitting wavelength become better.
The current direction and field of the study and application of molecular imaging include research of small animal imaging technique and development of new molecular probes, and research and study of imaging of in-vivo cell behavior and animal mode including gene expression, receptor and transporter, angiogenesis, drug resistance, drug abuse and targeted radionuclide therapy.
The subject was that after activation by an enzyme, a biocompatible near-infrared fluorescence imaging probe having light inhibition effect could generate a stronger signal and was designed into two kinds. One was a fluorescence supplier and the other was a quencher which was directly bound to two terminals of peptide substrate (G. Zlokarnik et al, Science, 1998, vol 279, page 84; S. V. Gulnik et al, FEBS LETT, 1997, vol 413, Page 379). The quencher was like to use a protected graft copolymer (PGC) which could help to inhibit a propagation of a tumor which was labeled by a near-infrared fluorescence probe and which was used in clinical test (R. Callahan et al, 1998, Am. J. Roentgenol. vol 171, page 137; C. H. Tung et al, 2000. Cancer Research, vol 60, page 4953).
A new near-infrared fluorescence (NIRF) probe generates an in-vivo image after activation by an enzyme. When the probe is not activated by the enzyme, the probe has self-quench effect and can not emit near-infrared light. When enzyme activates the probe, the self-quench effect diminishes and the probe can generate fluorescence light after activated by light.
The design for a general probe has several requisite properties, thus, the probe must has the following properties, such as, a longer recycle time, a high tumor accumulation. The probe was activated to emit a near-infrared fluorescence. The composition of the probe was formed by binding of an amino group at an upper portion of a poly-L-lysine (PL) backbone to methoxypoly(ethylene glycol) (MPEG) and binding of an amino acid sequences to an amino group at the other portion of the poly-L-lysine (PL) backbone with the other terminal of the amino acid sequences being bound to Cy5.5 dye so as to produce the so-called near-infrared fluorescence probe (see, U. Mahmood et al, 1999, Radiology, vol 213, page 866). In view of application of optical imaging to biological imaging, when an enzyme cut a near-infrared fluorescence probe, the probe was light-activated to generate a near-infrared fluorescence (NIR, λ=680-900 nm). Before the enzyme cut the probe, the probe had self-quench effect not to emit near-infrared fluorescence. Regarding in-vivo imaging, the probe provided relative superiority within near-infrared range (700-1000 nm). For example, water and most of the existing fluorochromes absorb the light energy within near-infrared range. Therefore, within infrared range emissions within near-infrared range it penetrated tissues more efficiently than that of visible light or photons. An ideal near-infrared fluorochrome for in-vivo imaging shall have the following features: (1) A peak of fluorescence shall be within 700-900 nm, (2) high quantum yield, (3) narrow excitation and emission spectrum, (4) chemical and light stability, (5) low toxicity, (6) biocompatibility, biodegradation and emission capability, (7) a few mono-functional derivatives can be obtained (8) commercial feasibility.
The images obtained by use of a probe with respect to different proteases were designed as disclosed in R. Weissleder et al, 2001, Nat. Med., vol 7, page 743. US 2003/0219383A1 disclosed two species of different peptides obtained and synthesized with respect to matrix metalloprotease 2 (MMP-2). One of the probe peptide structures was a peptide substrate Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys(FITC)-Cys-NH2 [SEQ ID NO: 1] with the other being a control peptide Gly-Val-Arg-Leu-Gly-Pro-Gly-Lys(FITC)-Cys-NH2 [SEQ ID NO: 2]. The cutting sites of the enzyme in U.S. Pat. application 2003/0219383A1 were (1) Lys●Lys, (2) PIC(Et)Phe●Phe, (3) His-Ser-Ser-Lys-Leu-Glne●[SEQ ID NO: 3], (4) Pro(Leu/Gln)Gly●(Ile/Lys)Arg-Gly [SEQ ID NO: 4], (5) Gly-Val-Val-Gin-Arge●Ser-Cys-Arg-Leu-Ala [SEQ ID NO: 5].
It was proved by a high performance liquid chromatography (HPLC) that an enzyme of matrix metalloprotease 2 (MMP-2) had the capability of cutting at Gly-Val residue while the control peptide could not be cut by the enzyme. The main purpose of an addition of fluorescein isothiocyanate (FITC) was used as a tag to measure amount of fluorescence and used to bind a thio group of cysteine of the peptide substrate to an amino group of a poly-L-lysine and to flurorchrome, to be used as a probe of matrix metalloprotease 2 (MMP-2).
A design of a probe for thrombin was disclosed in C.H. Tung et al, 2002, ChemBioChem., vol 3, page 207. One of the probe structures of the thrombin probe was a blood coagulation peptide substrate Gly-(D-Phe)-Pip-Arg-Ser-Gly-Gly-Gly-Gly-Lys(FITC)-Cys-NH2 while the other was a control peptide Gly-(D-Phe)-Pip-Arg-Pro-Gly-Gly-Gly-Gly-Lys(FITC)-Cys-NH2. By using a high performance liquid chromatography, it was judged and confirmed that the thrombin enzyme had capability of activate on Arg-Ser residue and that the control peptide had no activation effect on thrombin. It was disclosed in C.H. Tung et al., 2002, Angew. Chem., vol 114, page 3811 that a near--infrared fluorescence inhibitor of an azulene dimmer was synthesized and that a carboxyl group terminal was bound to a peptide sequence, i.e. Gly-Asp-Glu-Val-Asp-Gly-Ser-Gly-Cys [SEQ ID NO: 6] and the other Cys terminal was bound to a fluorochrome so as to emit near-infrared fluorescence. The peptide sequence could cut Asp-Glu-Val-Asp [SEQ ID NO: 7] residue by using capase-3.
It was disclosed in C. H. Tung et al, 2003, Tetrahedron Letters., vol 44, page 3975 a near-infrared quencher (NIRQ700) of mono-functional azulenyl squarain dye was synthesized and its wavelength absorption was within 600-700 nm. The wavelength absorption revealed that the probe could be effectively used as a quencher toward a fluorochrome within 600-750 nm.
The application of hormone-inhibitor (somatostatine) receptor to diagnosis and treatment was disclosed in K. Licha et al, 2001, Bioconjugate Chen., vol 12, page 44. A peptide-N-terminal amino functional group of a receptor-specific somatostatine was bound to an indodicarbocyanine (IDCC) and indotricarbocyanine (ITCC). The probe showed that it had a better molar absorption coefficient and fluorescence quantum yield. The results proved that the probe could emit near-infrared fluorescence and the probe was suitable for application as a probe of receptor-targeted molecular imaging.
It was disclosed in A. Becke et al, 2001, Nature Biotechnology, vol 19, page 327 that a cyanine dye derivative was bound to a hormone receptor antagonist (octreotate) peptide derivative useful as a probe of optical imaging. Its in-vivo images showed that the peptide effect was best when indodicarbocyanine (IDCC) was bound to hormone receptor antagonist (octreotate).
It was disclosed in C. H. Tung et al, 2002, ChemBioChem, vol 8, page 784 that a small molecular probe was used to emit near-infrared fluorescence (NIRF: 700-900 nm), for the targeted compound of fluorescence molecular imaging the small molecular probe was especially bound to a peptide and a fluorochrome. The results demonstrated that in the in-vivo optical image the probe was targeted to folate receptor in order to detect many forms of tumors, especially ovarian cancer. Therefore, the small molecular probe had a better medicine kinetics and non-immunity property.
From the results of the above literatures, it was shown that probes were designed to be used on various proteases. In the present invention, an optical imaging probe obtained by binding a peptide to near-infrared fluorochrome (Cy5.5) to form a bioconjugate. The present invention provides a prostate peptide substrate designed for a prostate-specific antigen enzyme, which is Gly-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Met-Lys(FITC)-Cys-NH2 [SEQ ID NO: 8]. Methoxypoly(ethylene glycol) (MPEG) is bound to an amino group at upper portion of poly-L-lysine (PL) and two terminals of the designed amino acid sequences are respectively bound to poly-L-lysine (PL) and Cy5.5 dye to form a near-infrared fluorescence probe. The specificity of the prostate-specific antigen enzyme is studied.
The cyclohexyl-gly (Chg) is an amino acid having the following formula:
